The present invention concerns new compounds for inhibiting viral fusion.
“Enveloped viruses”, such as orthomyxoviruses, paramyxoviruses, retroviruses, flaviviruses, rhabdoviruses and alphaviruses, are surrounded by a lipid bilayer originating from the host plasma membrane (Ono and Freed, Adv. Virus Res., 2005, 273, 5419-5442). This envelope contains glycoproteins that mediate receptor binding and fusion between viral and host cell membranes. Cholesterol and sphingomyelin are often enriched in these viral lipid bilayers, particularly in lipid-rich rafts in their plasma membrane (Aloia et al, PNAS, 1988, 85, 900-4 and 1993, 90, 5181-5).
In HIV-1 the surface glycoprotein, initially synthesized as a highly glycosylated precursor, gp160, is endoproteolytically cleaved into the surface protein, gp120, that determines the viral tropism through the cellular surface receptors, and the transmembrane protein, gp41, which is responsible for the membrane fusion process (Moreno et al, Biochimica et Biophysica Acta, 2006, 1758, 111-123).
Cholesterol comprises about 30% of the lipid content of the plasma membrane of mammalian cells. Cholesterol and sphingolipids in membranes are often laterally segregated from surrounding glycerolipid-rich bilayers to form membrane microdomains known as “lipid rafts” (P. Casey, Science, 1995, 268, 221-5). A number of transmembrane proteins and receptors, including CD4 which is the primary receptor for HIV envelope gp120, are particularly enriched in lipid rafts. To accomplish the fusion and mixing of cellular and viral contents, gp41 must undergo a complex series of conformational changes apparently triggered by the attachment of gp120 to the CD4 primary receptor and the CCR5 or CXCR4 coreceptors of the target cell.
The completion of co-receptor binding leads to the fusion-active conformation of the viral transmembrane fusion protein gp41. The ectodomain of gp41 contains two heptad repeat regimes: HR1 (proximal to the N terminus) and HR2 (proximal to the C terminus). The hydrophobic fusion peptide region inserts into the host cell membrane, whereas the HR1 region of gp41 form a trimeric coiled coil structure. HR2 regions then fold back within the hydrophobic grooves of the HR1 coiled coil, forming a hairpin structure containing a thermodynamically stable six-helix bundle that draws the viral and cellular membranes together for fusion (Matthews et al, Nature Reviews Drug Discovery, 2004, 3, 215-225).
It has also been shown that gp41 associates with caveolin-1, a structural protein component of a subset of lipid rafts called ‘caveolae’ (Hovanession et al, Immunity, 2004, 21, 617-627). Caveolin-1 is a cholesterol-binding protein (Murata et al, PNAS, 1995, 92, 10339-10343), so cholesterol is enriched in caveolae, together with HIV-1.
Whatever the precise mechanism, a substantial body of evidence supports the importance of lipid rafts and cholesterol in enveloped virus entry and, for HIV at least, it is generally thought that the lipid rafts on host cell plasma membranes play an essential role in mediating gp120/CD4/coreceptor interactions, while the cholesterol/lipid rafts in HIV viral lipid bilayers are important for maintaining normal structure and function of viral glycoproteins and hence viral infectivity (Ono and Freed, supra).
Proteins containing lipid anchors are commonly found. One type, which has been described recently for Hedgehog proteins, involves a cholesterol molecule esterified to the C-terminal amino acid of a protein following a self-splicing reaction (Tanaka Hall et al, Cell, 1997, 91, 85-97).
The relative ability of lipid anchors to stably localise their associated proteins to lipid membranes has been extensively studied. There is a general relationship between the degree of hydrophobic modification and the stability of membrane insertion: quasi-irreversible binding requires the presence of two long chain anchors in the molecule, for example palmitoyl and farnesyl, or hexadecyl and farnesyl (Barder et al, Nature, 2000, 403, 223-226). By contrast, quasi-irreversible binding is achieved with a single cholesterol moiety (Peters et al, PNAS, 2004, 101, 8531-6). Notably, this modification is effective for a protein (N-Ras) which is normally anchored via a different lipid.
A general advantage of targeting a peptide to a membrane is to effectively increase its concentration over the bulk aqueous phase. This results in augmenting its binding affinity toward membrane-bound receptors.
For antiviral agents, including peptides, proteins, and antibodies, which target fusion as mechanism of action, a number of examples document the advantage of anchoring the inhibitor to a membrane. For HIV in particular, a construct, including the fusion inhibitor T20 (enfuvirtide, FUZEON®) (Matthews et al, supra), a short linker and a transmembrane (TM) domain, was a much more powerful inhibitor than the same construct lacking the TM domain (Egelhofer et al, J. Virol., 2004, 78, 568-575). Importantly, mutations in the Trp-rich region which completely inactivated the free peptide, did not reduce the potency of the membrane-anchored one (Hildinger et al, J. Virol., 2001, 75, 3038-3042). Similarly a construct including the TM domain of gp41 and the entire C-terminal heptad repeat HR2 incorporated into liposomes had potent antiviral activity (Lenz et al, J. Biol Chem., 2004, 280, 4095-4101).
Addition of a C-terminal octyl group to the fusion inhibitor T20 induced a significant increase in its inhibitory potency. Furthermore, octylation could rescue the activity of an otherwise inactive mutant, in which the C-terminal residues GNWF were replaced by ANAA. The mutant with a C-terminal octyl group showed potency similar to that of the wild type T20. Importantly, the position of the octyl group was critical, since N-terminal derivatization had no effect on antiviral potency (Peisajovich et al, J. Biol. Chem., 2003, 278, 21012-7). An increased ability to partition into membranes has been recently proposed as the reason behind the increased clinical efficacy of the 2nd-generation inhibitor T1249 when compared with T20 (Veiga et al, J. Am. Chem. Soc, 2004, 126, 14758-63).
N-terminal extension of the chemokine RANTES, a natural ligand of the HIV co-receptor CCR5, with a hydrophobic group, has been used to greatly increase antiviral potency. Both the hydrophobicity and the chemical nature of the connection with the protein were important for maximal potency (Mosier et al, 1999, J. Viral, 1999, 73, 3544-50).
It has also been shown that when a bona fide non-neutralizing antibody, which did not inhibit HIV-1 entry when produced as a soluble protein, was anchored to the cell surface of target cell by fusion with a transmembrane anchoring domain, it acted as a neutralizing antibody (Lee et al, J. Immunol., 2004, 173, 4618-26).